Progress towards an HIV-1 vaccine has been stymied by the inability to induce a protective humoral response. Well-characterized neutralization epitopes are either poorly immunogenic (e.g., b12, 2F5, 2G12) or effectively masked on the majority of primary isolates (e.g., antibodies to V3, CD4-bd and CD4-i epitopes). New evidence suggests that even highly masked primary isolates possess sensitive neutralization targets that are recognized by autologous patient sera and occasionally by heterologous sera. This suggests that mapping the epitopes involved may identify novel targets that are capable of inducing broadly neutralizing activities. We have identified several patient sera that possess broadly neutralizing activities for primary isolates, and we have developed methods for localizing the target epitopes. We now propose to identify epitopes that mediate neutralization of both autologous and heterologous clade B and clade C Envs. The Specific Aims are: 1- To screen a large number of patient sera obtained from both North-American and African cohorts for cross-neutralizing activities, and select samples with broad neutralizing properties for detailed characterization. 2- To map target epitopes initially by examining the neutralization sensitivities of chimeras formed between neutralization-sensitive and -resistant Envs, in which key domains were exchanged using available or engineered restriction sites. Finer mapping of the epitopes involved will be performed by exchanging smaller regions and by mutagenesis of key residues. In parallel, sensitive epitopes will be mapped immunochemically using gp120 and gp41 antigens and fusion proteins expressing various domains of sensitive Envs as blocking and immunoadsorption reagents in neutralization assays. 3- To isolate monoclonal antibodies (mAbs) with broad neutralizing activities from EBV-transformed B cell cultures obtained from these patients, and from mice immunized with Env proteins or fusion proteins expressing potential neutralization targets. Screening will be performed both with binding and direct neutralization assays. The resulting mAbs will be used to further define the structure and distribution of the epitopes involved. 4- Finally, to immunize rabbits with fusion proteins expressing novel targets. These studies will evaluate the immunogenicity of the expressed sequences and to test the neutralizing properties of antibodies induced against these immunogens. It is anticipated that these studies will define novel targets that are exposed on typical neutralization-resistant primary isolates and responsible for broad neutralization, and could lead to new vaccine approaches based on immunogens expressing combinations of such epitopes.